Changes In Mitochondrial Metabolism Research Articles

Abstract 4115015: Hyperpolarized Carbon-13 Metabolic Imaging Detects Changes in Cardiac Mitochondrial Metabolism in Patients Before and After Coronary Artery Bypass Graft Surgery

Background: Coronary Artery Disease (CAD) is a significant global health issue, necessitating improved diagnostic tools for visualizing cardiac energetics. Traditional imaging methods such as PET and dobutamine stress echocardiography do not directly assess mitochondrial metabolism. Hyperpolarized Carbon-13 metabolic magnetic resonance imaging (HP- 13 C MRI) offers a promising non-invasive method for investigating mitochondrial function in CAD. This study utilizes HP- 13 C MRI to detect changes in mitochondrial metabolism in patients undergoing Coronary Artery Bypass Graft (CABG) surgery (Fig.1). Methods: We conducted HP- 13 C MRI examinations on two patients with advanced CAD before and (~4-6 months after CABG surgery and one healthy subject (Fig. 2 A,B). Participating subjects provided informed consent according to a protocol approved by the Institutional Review Board and Protocol Review Committee. Baseline blood samples were analyzed for pyruvate, triglycerides, free fatty acids, and insulin levels. Post-glucose load, patients received an intravenous injection of an IND-approved metabolic probe, [1- 13 C] pyruvic acid, prepared under Good Manufacturing Practice regulations. The HP solution was administered after polarization in a clinical polarizer (SPINlab™, GE Healthcare). Imaging was performed using a GE MR750 MR system with a Helmholtz loop-pair 13 C coil (PulseTeq Limited, UK). Data were reconstructed and analyzed with MATLAB scripts. Results and Discussion: Baseline blood measurements were normal for the healthy subject, but those with advanced CAD showed variable and abnormal values (Fig. 2C). HP- 13 C MRI safely assessed cardiac metabolism in patients with advanced CAD. Patients with advanced CAD exhibited reduced pyruvate metabolism compared to healthy controls, shown by lower myocardial bicarbonate/(bicarbonate+lactate) ratios (Bic/(Bic+Lac)). Following CABG, only Patient 2 showed improved Bic/(Bic+Lac), while Patient 1 did not (Fig. 3A-B). This variability may be influenced by differences in nutrition, hormonal status, medication regimens, or other factors. Changes in % Bic/(Bic+Lac) across different coronary artery segments were observed post-CABG in CAD patients (Fig. 3C). Conclusion: HP- 13 C MRI non-invasively assesses cardiac metabolism in CAD patients, demonstrating the potential to evaluate post-CABG metabolic changes. Our efforts continue to recruit large cohort to understand individual variability.

New insights into the pathophysiology of rheumatic valve disease based on different genetic and proteomics profiles

Abstract Introduction Rheumatic heart disease (RHD) pathogenesis is not fully understood. Purpose The protein profile in rheumatic heart disease may have a different fingerprint in the aortic and mitral valves, which results in different evolution in terms of valve damage. Methods To evaluate the genetic profile in symptomatic RHD, 4 simultaneous mitral and aortic valves were obtained during valve replacement, as well as 4 "normal" valves from cadavers (Control Group). The expression of 90 genes was evaluated using a customized RT-PCR array kit. A second experiment was carried out to evaluate proteomics. Then, 7 mitral and aortic valves, from the same patient, were also collected during valve surgery. The Control Group consisted of 6 mitral and aortic valves obtained from explanted hearts during heart transplantation due to non-valvular pathologies. Additionally, histopathological and sectoral proteomic analysis of amyloid deposits, found in some mitral and aortic valves, from the second experiment, were also reported. Results Mitral rheumatic valves exhibited higher expression of genes linked to calcification and immuno-inflammation compared to aortic rheumatic valves (Figure 1). Proteomic analysis, visualized through PCA graphs, highlighted differences in proteomic patterns. Despite having the same genetic pattern, control valves exhibited different proteomic profiles (Figure 1) Rheumatic mitral and aortic valves, even from the same individual, showed distinct proteomic fingerprint (Figure 1). A total of 49 significant proteins were identified in distinguishing healthy mitral and aortic valves from the same individual (Figure 1). The aortic valve displayed a richer extracellular matrix and higher levels of housekeeping proteins that maintain valve homeostasis compared to the mitral valve (Figure 1). The dominant proteomic pattern in valvular rheumatic disease was identified as alterations in critical mitochondrial metabolic activities related to proteostasis. (Figure 2). Particularly notable was the upregulation of pathways associated with lipid metabolism (Figure 2). Finally, amyloid deposits suggested APOA1 as a potential deposit protein in RHD (Figure 2). Conclusion The study confirms distinct constitutional differences between mitral and aortic valves affecting rheumatic disease progression. It emphasizes the role of altered proteostasis and altered proteins deposit, mitochondrial metabolic changes, and inflammatory responses in the disease's pathophysiology, highlighting the complexity of valvular rheumatic disease involvement.For the first time amyloid deposition has been reported as a likely consequence of significant disruption in valvular proteostasis and the proteins potentially comprising this deposit within a rheumatic valve.Genomic and proteomic overviewproteomic fingerprint of RHD and amyloid

Misprogramming of glucose metabolism impairs recovery of hippocampal slices from neuronal GLT-1 knockout mice and contributes to excitotoxic injury through mitochondrial superoxide production.

We have previously reported a failure of recovery of synaptic function in the CA1 region of acute hippocampal slices from mice with a conditional neuronal knockout (KO) of GLT-1 (EAAT2, Slc1A2) driven by synapsin-Cre (synGLT-1 KO). The failure of recovery of synaptic function is due to excitotoxic injury. We hypothesized that changes in mitochondrial metabolism contribute to the heightened vulnerability to excitotoxicity in the synGLT-1 KO mice. We found impaired flux of carbon from 13C-glucose into the tricarboxylic acid cycle in synGLT-1 KO cortical and hippocampal slices compared with wild-type (WT) slices. In addition, we found downregulation of the neuronal glucose transporter GLUT3 in both genotypes. Flux of carbon from [1,2-13C]acetate, thought to be astrocyte-specific, was increased in the synGLT-KO hippocampal slices but not cortical slices. Glycogen stores, predominantly localized to astrocytes, are rapidly depleted in slices after cutting, and are replenished during exvivo incubation. In the synGLT-1 KO, replenishment of glycogen stores during exvivo incubation was compromised. These results suggest both neuronal and astrocytic metabolic perturbations in the synGLT-1 KO slices. Supplementing incubation medium during recovery with 20 mM D-glucose normalized glycogen replenishment but had no effect on recovery of synaptic function. In contrast, 20 mM non-metabolizable L-glucose substantially improved recovery of synaptic function, suggesting that D-glucose metabolism contributes to the excitotoxic injury in the synGLT-1 KO slices. L-lactate substitution for D-glucose did not promote recovery of synaptic function, implicating mitochondrial metabolism. Consistent with this hypothesis, phosphorylation of pyruvate dehydrogenase, which decreases enzyme activity, was increased in WT slices during the recovery period, but not in synGLT-1 KO slices. Since metabolism of glucose by the mitochondrial electron transport chain is associated with superoxide production, we tested the effect of drugs that scavenge and prevent superoxide production. The superoxide dismutase/catalase mimic EUK-134 conferred complete protection and full recovery of synaptic function. A site-specific inhibitor of complex III superoxide production, S3QEL-2, was also protective, but inhibitors of NADPH oxidase were not. In summary, we find that the failure of recovery of synaptic function in hippocampal slices from the synGLT-1 KO mouse, previously shown to be due to excitotoxic injury, is caused by production of superoxide by mitochondrial metabolism.

Simultaneous Second Harmonic Generation and Multiphoton Excited Photoluminescence in Samarium-Doped BaTiO3 Nanoparticles Functionalized with Poly(ethylene glycol).

In this work, samarium-doped BaTiO3 (BT:Sm) nanoparticles (NPs) were prepared and coated with poly(ethylene glycol) (PEG) to investigate their optical characteristics and compatibility with biological systems. The structure, particle morphology, optical properties, and biological compatibility of the NPs were assessed. The results demonstrated the formation of BT:Sm and [(BT:Sm)-PEG]. The relative intensities and positions of peaks in the X-ray diffraction (XRD) are consistent with an average crystallite size of ∼75 nm. The Raman spectra showed that Sm doping produced the typical tetragonal peaks at around 306 and 715 cm-1, and Fourier transform infrared (FTIR) spectroscopy showed that the PEGylation process was effective. Also, our investigation demonstrates the potential of these NPs as very temperature-sensitive nanosensors with a resolution exceeding 0.5 °C, which is achievable through optical excitation. We also analyze their emission properties. Finally, we present a study related with the mitochondrial activity of naked and PEG-coated NPs. The results indicate that neither naked nor PEG-coated NPs exhibit changes in mitochondrial metabolism, as indicated by quantitative cell viability and morphological visualization. The PEG-coated NPs prevented the formation of aggregates in cell culture compared to naked NPs, demonstrating the significance of PEG as a stabilizing agent.

Mitochondrial metabolism regulation and epigenetics in hypoxia.

The complex and dynamic interaction between cellular energy control and gene expression modulation is shown by the intersection between mitochondrial metabolism and epigenetics in hypoxic environments. Poor oxygen delivery to tissues, or hypoxia, is a basic physiological stressor that sets off a series of reactions in cells to adapt and endure oxygen-starved environments. Often called the "powerhouse of the cell," mitochondria are essential to cellular metabolism, especially regarding producing energy through oxidative phosphorylation. The cellular response to hypoxia entails a change in mitochondrial metabolism to improve survival, including epigenetic modifications that control gene expression without altering the underlying genome. By altering the expression of genes involved in angiogenesis, cell survival, and metabolism, these epigenetic modifications help cells adapt to hypoxia. The sophisticated interplay between mitochondrial metabolism and epigenetics in hypoxia is highlighted by several important points, which have been summarized in the current article. Deciphering the relationship between mitochondrial metabolism and epigenetics during hypoxia is essential to understanding the molecular processes that regulate cellular adaptation to reduced oxygen concentrations.

FAM83B regulates mitochondrial metabolism and anti-apoptotic activity in pulmonary adenocarcinoma.

Chemotherapy is an effective therapeutic modality; nevertheless, a significant proportion of patients diagnosed with lung adenocarcinoma (LUAD) demonstrate resistance to chemotherapy. Therefore, it is crucial to understand the potential regulatory mechanisms to develop novel treatment strategies. This study aims to understand how increased FAM83B expression impacts mitochondrial activity, cell apoptosis, and chemotherapy effectiveness in LUAD. Multiple assays, such as CCK8, wound healing, EdU, and transwell assays, were employed to confirm the augmented chemotherapy resistance, heightened cell proliferation, migration, and invasion caused by FAM83B overexpression in LUAD cells. Furthermore, MIMP, MTG, and ATP assays were utilized to quantify changes in mitochondrial metabolism. In vitro functional assays were performed to evaluate the influence of FAM83B overexpression on the malignant progression and resistance mechanisms to chemotherapy in LUAD. In the context of this study, it was determined that LUAD patients with increased FAM83B expression had shorter survival times, and tissue samples with FAM83B overexpression were more prone to metastasis compared to primary samples. As a result, FAM83B is identified as an adverse prognostic marker. The mechanistic analysis demonstrated that FAM83B impedes the translocation of calbindin 2 (CALB2) from the cytoplasm to the mitochondria, resulting in the inhibition of apoptosis and the promotion of mitochondrial activity. Consequently, this ultimately confers resistance to chemotherapy in LUAD. Furthermore, the administration of metformin, which blocks mitochondrial oxidative phosphorylation (OXPHOS), can restore sensitivity to drug resistance in LUAD.Taken together, these findings provide substantial evidence supporting the notion that FAM83B enhances chemotherapy resistance in LUAD through the upregulation of mitochondrial metabolism and the inhibition of apoptosis.

Breast Cancer: Mitochondria-Centered Metabolic Alterations in Tumor and Associated Adipose Tissue.

The close cooperation between breast cancer and cancer-associated adipose tissue (CAAT) shapes the malignant phenotype, but the role of mitochondrial metabolic reprogramming and obesity in breast cancer remains undecided, especially in premenopausal women. Here, we examined mitochondrial metabolic dynamics in paired biopsies of malignant versus benign breast tumor tissue and CAAT in normal-weight and overweight/obese premenopausal women. Lower protein level of pyruvate dehydrogenase and citrate synthase in malignant tumor tissue indicated decreased carbon flux from glucose into the Krebs cycle, whereas the trend was just the opposite in malignant CAAT. Simultaneously, stimulated lipolysis in CAAT of obese women was followed by upregulated β-oxidation, as well as fatty acid synthesis enzymes in both tumor tissue and CAAT of women with malignant tumors, corroborating their physical association. Further, protein level of electron transport chain complexes was generally increased in tumor tissue and CAAT from women with malignant tumors, respective to obesity. Preserved mitochondrial structure in malignant tumor tissue was also observed. However, mitochondrial DNA copy number and protein levels of PGC-1α were dependent on both malignancy and obesity in tumor tissue and CAAT. In conclusion, metabolic cooperation between breast cancer and CAAT in premenopausal women involves obesity-related, synchronized changes in mitochondrial metabolism.

The role of cardiolipin and cytochrome c in mitochondrial metabolism of cancer cells determined by Raman imaging: in vitro study on the brain glioblastoma U-87 MG cell line.

In this paper, we present Raman imaging as a non-invasive approach for studying changes in mitochondrial metabolism caused by cardiolipin-cytochrome c interactions. We investigated the effect of mitochondrial dysregulation on cardiolipin (CL) and cytochrome c (Cyt c) interactions for a brain cancer cell line (U-87 MG). Mitochondrial metabolism was monitored by checking the intensities of the Raman bands at 750 cm-1, 1126 cm-1, 1310 cm-1, 1337 cm-1, 1444 cm-1 and 1584 cm-1. The presented results indicate that under pathological conditions, the content and redox status of Cyt c in mitochondria can be used as a Raman marker to characterize changes in cellular metabolism. This work provides evidence that cardiolipin-cytochrome c interactions are crucial for mitochondrial energy homeostasis by controlling the redox status of Cyt c in the electron transport chain, switching from disabling Cyt c reduction and enabling peroxidase activity. This paper provides experimental support for the hypothesis of how cardiolipin-cytochrome c interactions regulate electron transfer in the respiratory chain, apoptosis and mROS production in mitochondria.

Modulation of Mitochondrial Dynamics by the Angiotensin System in Dopaminergic Neurons and Microglia.

Renin-angiotensin system (RAS) dysfunctions have been associated to life-spam, and aging-related diseases, including neurodegenerative diseases, such as Parkinson's disease, and the neuroinflammatory associated processes. Mitochondrial dysfunctions play a major role in aging-related diseases, including dopaminergic neurodegeneration and neuroinflammation. However, the mechanisms of RAS/mitochondria interactions remain to be clarified. In the present work, we studied the role of major RAS components in the mitochondrial dynamics in dopaminergic neurons and microglia using in vitro and in vivo models. In dopaminergic neurons, we observed that activation of the RAS pro-oxidative/pro-inflammatory axis (Angiotensin II/Angiotensin type-1 receptor, AT1/NADPH oxidase complex) produces a dysregulation of mitochondrial dynamics towards mitochondrial fission, via Drp1 phosphorylation at Ser616 and translocation to mitochondria. However, activation of the RAS antioxidative/anti-inflammatory axis, using Angiotensin 1-7, counteracts this effect. RAS components also modulated the microglial inflammatory response through mitochondrial dynamic changes. After interferon-γ-induced activation of human microglial cells, we observed increased mitochondrial fission and superoxide production that was inhibited by Angiotensin 1-7 treatment. Angiotensin 1-7 also inhibited mitochondrial metabolic changes induced by pro-inflammatory microglial activation. The role of RAS in mitochondrial dynamic changes was confirmed in vivo using the LPS-induced inflammation model in wild-type, AT1-KO, and AT2-KO mice. The effect of Angiotensin 1-7 is mediated by IL-10, specifically by decreasing the post-transcriptional phosphorylated Drp1 form, and translocation of STAT3 to mitochondria. Angiotensin 1-7, acting on mitochondrial Angiotensin 1-7 receptors (Mas/Mas related receptors), increased the phosphorylated form of STAT3 at Ser727, which is mediated by mitochondrial PKA activation. In conclusion, the present findings show the role of RAS components in modulation of mitochondrial dynamics and mitochondrial function, revealing the associated signaling pathways. The results lead to better understanding of the effects of RAS dysfunction in aging-related diseases, and particularly dopaminergic degeneration and neuroinflammation in Parkinson's disease.

Mitochondrial Mechanisms of Endothelial Cell Damage in Alzheimer’s Disease and Cerebral Amyloid Angiopathy

AbstractBackgroundCerebrovascular endothelial cells (EC) are crucial regulators of cerebral homeostasis and function through the maintenance of the blood‐brain barrier (BBB). ECs damage and/or dysfunction may result in defects in brain clearance and perfusion, microhemorrhages, inflammation, and neurodegeneration. Damage to the ECs may result from cardiovascular risk factors (CVRF) such as high levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy). In Alzheimer’s Disease (AD) and Cerebral Amyloid Angiopathy (CAA), the presence of a CVRF may further exacerbate cerebrovascular function and neurodegeneration. EC do not rely highly on mitochondrial ATP production as they are glycolytic cells. Instead, EC mitochondria serve mostly as signaling organelles, which sense cellular damage and mediate downstream pathways such as inflammation. In this study, we aim to understand the mechanistic nature of endothelial mitochondrial dysfunction and its elicited pathways in Aβ‐ and mixed (Aβ+HHcy)‐mediated vascular pathologies. Understanding the mechanisms by which the mitochondria mediate EC inflammation, may pave the way to new roads for therapeutic advancements in the field.MethodHuman brain microvascular ECs were challenged with Aβ, Hcy, or the combination. Mitochondrial metabolic function, inflammatory cascades and signals, and EC barrier function were assessed.ResultThe presence of Aβ, but not Hcy, induced deficits in mitochondrial respiration and ATP production. In addition, there is evidence of mitochondrial fragmentation and the expression of fission proteins. Preliminary results further suggest a release of mitochondrial danger associated molecular patterns (mtDAMPs) that can promote an inflammatory cascade. The mitochondrial damage and subsequent inflammatory response are associated with an increase in EC barrier permeability and cytokine release, which is exacerbated in the presence of Hcy.ConclusionThe EC dysfunction associated with Aβ is accompanied by severe deficits in mitochondrial metabolism and the release of mtDAMPS. The presence of Hcy exacerbates some of the detrimental effects of Aβ, particularly on mtDAMPS and BBB function, but Hcy does not appear to affect mitochondrial metabolic changes. Overall, our results reveal a novel role for mtDAMPs in vascular dysfunction in the presence of Aβ and/or Hcy.

Reduced Mitochondrial Protein Translation Promotes Cardiomyocyte Proliferation and Heart Regeneration.

The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation. We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed. We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

Mitochondrial regulation in human pluripotent stem cells during reprogramming and β cell differentiation.

Mitochondria are the powerhouse of the cell and dynamically control fundamental biological processes including cell reprogramming, pluripotency, and lineage specification. Although remarkable progress in induced pluripotent stem cell (iPSC)-derived cell therapies has been made, very little is known about the role of mitochondria and the mechanisms involved in somatic cell reprogramming into iPSC and directed reprogramming of iPSCs in terminally differentiated cells. Reprogramming requires changes in cellular characteristics, genomic and epigenetic regulation, as well as major mitochondrial metabolic changes to sustain iPSC self-renewal, pluripotency, and proliferation. Differentiation of autologous iPSC into terminally differentiated β-like cells requires further metabolic adaptation. Many studies have characterized these alterations in signaling pathways required for the generation and differentiation of iPSC; however, very little is known regarding the metabolic shifts that govern pluripotency transition to tissue-specific lineage differentiation. Understanding such metabolic transitions and how to modulate them is essential for the optimization of differentiation processes to ensure safe iPSC-derived cell therapies. In this review, we summarize the current understanding of mitochondrial metabolism during somatic cell reprogramming to iPSCs and the metabolic shift that occurs during directed differentiation into pancreatic β-like cells.

Spectroscopic imaging to assess biochemical alterations in liver carcinoma cells exposed to transition metals

Despite the invaluable role of transition metals in every living organism, it should be remembered that failure to maintain the proper balance and exceed the appropriate dose may have the opposite effect. In the era of such a popular and propagated need for supplementation in the media, one should bear in mind the harmful effects that may become the result of improper and excessive intake of transition metals.This article establishes the feasibility of Raman (RS) and Fourier-transform infrared (FT-IR) spectroscopic imaging at the single-cell level to investigate the cellular response to various transition metals. These two non-destructive and perfectly complementary methods allow for in-depth monitoring of changes taking place within the cell under the influence of the agent used. HepG2 liver carcinoma cells were exposed to chromium, iron, cobalt, molybdenum, and nickel at 1 and 2 mM concentrations.Spectroscopic results were further supported by biological evaluation of selected caspases concentration. The caspase- 3, 6, 8, 9, and 12 concentrations were determined with the use of the enzyme-linked immunosorbent assay (ELISA) method. This study shows the induction of apoptosis in the intrinsic pathway by all studied transition metals. Cellular metabolism alterations are induced by mitochondrial metabolism changes and endoplasmic reticulum (ER) metabolism variations. Moreover, nickel induces not only the intrinsic pathway of apoptosis but also the extrinsic pathway of this process.

Clofazimine-Mediated, Age-Related Changes in Skeletal Muscle Mitochondrial Metabolites.

Mitochondrial health declines with age, and older patients can demonstrate dysfunction in mitochondrial-rich tissues, such as cardiac and skeletal muscle. Aged mitochondria may make older adults more susceptible to adverse drug reactions (ADRs). We assessed mitochondrial metabolic function by measuring two metabolites, l-carnitine and acetylcarnitine, to determine their effectiveness as candidate clinical biomarkers for age-related, drug-induced alterations in mitochondrial metabolism. To study age- and medication-related changes in mitochondrial metabolism, we administered the FDA-approved mitochondriotropic drug, clofazimine (CFZ), or vehicle for 8 weeks to young (4-week-old) and old (61-week-old) male C57BL/6J mice. At the end of treatment, whole blood and cardiac and skeletal muscle were analyzed for l-carnitine, acetylcarnitine, and CFZ levels; muscle function was measured via a treadmill test. No differences were found in blood or cardiac carnitine levels of CFZ-treated mice, but CFZ-treated mice displayed lost body mass and alterations in endurance and levels of skeletal muscle mitochondrial metabolites. These findings demonstrate the age-related susceptibility of the skeletal muscle to mitochondria drug toxicity. Since drug-induced alterations in mitochondrial metabolism in skeletal muscle were not reflected in the blood by l-carnitine or acetylcarnitine levels, drug-induced catabolism and changes in muscle function appear more relevant to stratifying individuals at increased risk for ADRs.

Spatiotemporal Coordination of RPE Cell Quality by Extracellular Vesicle miR-494-3p Via Competitive Interplays With SIRT3 or PTEN.

To reveal the molecular mechanism underlying degeneration in human retinal pigment epithelial (hRPE) cells with dysfunctional mitochondrial homeostasis. The expression of recently identified miR-494-3p in extracellular vesicles (EV) released from induced-pluripotential-stem-cell-derived human RPE (iPS-hRPE), during coculture with macrophages (Mps) was investigated in iPS-hRPE and ARPE cells differentiated in the presence of nicotinamide (Nic-ARPE). The expression of phosphatase and tensin homolog (PTEN), sirtuin3 (SIRT3), and mitochondrial marker proteins before and after the transfection of miR-494-3p inhibitor and mimic, and the changes in mitochondrial metabolism, membrane potential, and oxidative phosphorylation (OXPHOS) were monitored. Compared with senescent dedifferentiated ARPE19 cells, iPS-hRPE and Nic-ARPE cells expressed elevated levels of mitochondrial marker proteins but a repressed cellular miR-494-3p level. The expression of target proteins of miR-494-3p, PTEN, and SIRT3 was upregulated along with the differentiation disposition of these RPE cells. The ratio of PTEN/SIRT3 in de-differentiated ARPE19 cells was surprisingly elevated by around 20 times compared with that in iPS-hRPE and Nic-ARPE cells. The novel molecular interplay of EV miR-494-3p either with mitochondria selective SIRT3 or organelle nonselective PTEN was found to participate in the degeneration of hRPE cells by inducing mitochondrial dysfunctions and repressed OXPHOS, mitochondrial membrane potential, and ATP and NAD+ production. Our results demonstrate a clear causal link between miR-494-3p and hRPE cell degeneration via the regulation of mitochondrial integrity. EV miR-494-3p may play a pivotal role in pathogenic spreading of degenerated hRPE cells from the local perifovea throughout the macula.

PO-02-124 MITOCHONDRIAL GLYCEROL-3-PHOSPHATE DEHYDROGENASE ACTS AT THE CROSSROAD OF METABOLISM REPROGRAMMING IN NON-VALVULAR ATRIAL FIBRILLATION: INDICATION FROM MULTI-OMICS ANALYSIS OF HUMAN AF MYOCARDIAL TISSUE

Metabolic remodeling has been known to play an important role in the development of atrial fibrillation(AF). Recent studies have shown prominent mitochondrial metabolic changes of atrium, which have significant impact on the electrical signaling and structural remodeling of atrium. However, there is limited data on AF metabolic remodeling from multi-omics analysis of human myocardial tissue. In addition, the metabolism-related hub gene and pathways are not completely known. We aimed to explore the mitochondrial metabolism reprogrammed in AF, and describe the key dysregulated molecules of AF patients, as well as related metabolic pathways. In-hospital patients diagnosed with AF from 2019 to 2022 were enrolled. Left atrial appendages were harvested from AF patients undergoing cardiovascular surgery and matched ones from healthy donors with sinus rhythm(SR). Metabolomics and proteomics were then performed and analyzed. Moreover, biostatistical analysis was done for further identification. Heart tissue samples were harvested from 10 persistent non-valvular AF patients (Table1) and 10 matched SR samples from healthy donors. In metabolomics analysis, 113 metabolites were up-regulated and 10 were down-regulated (Panel A), where multiple pathways related to amino acid and glycolipid metabolism were enriched (Panel B). After correlation analysis between the differentially expressed proteins and metabolites, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) was found crucial in AF patients in both positive- and negative-ionized mode (MCC score=16 and MCC score=6 respectively)(Panel C-D), which locates in the inner membrane of mitochondria and participates in cardiac mitochondrial function and glucolipid homeostasis. Proteomics revealed that the mitochondrial metabolism demonstrated marked difference in AF hearts than SR, especially the oxidative phosphorylation (NES=-1.73)(Panel E), ATP biosynthetic process (NES:-2.29), NADH dehydrogenase (NES:-2.36) etc., which were closely related to the function of mGPDH. The expression level of mGPDH was associated with multiple pathways related to cardiac structural remodeling using gene set variation analysis, which was further validated by external database (Panel F). Mitochondrial metabolism underwent prominent reprogramming in AF. mGPDH may participate in the development of AF via regulating mitochondrial metabolism and contribute to atrial remodeling, which could be considered as a a novel target for AF treatment.

Reverse Electron Transport at Mitochondrial Complex I in Ischemic Stroke, Aging, and Age-Related Diseases.

Stroke is one of the leading causes of morbidity and mortality worldwide. A main cause of brain damage by stroke is ischemia-reperfusion (IR) injury due to the increased production of reactive oxygen species (ROS) and energy failure caused by changes in mitochondrial metabolism. Ischemia causes a build-up of succinate in tissues and changes in the mitochondrial NADH: ubiquinone oxidoreductase (complex I) activity that promote reverse electron transfer (RET), in which a portion of the electrons derived from succinate are redirected from ubiquinol along complex I to reach the NADH dehydrogenase module of complex I, where matrix NAD+ is converted to NADH and excessive ROS is produced. RET has been shown to play a role in macrophage activation in response to bacterial infection, electron transport chain reorganization in response to changes in the energy supply, and carotid body adaptation to changes in the oxygen levels. In addition to stroke, deregulated RET and RET-generated ROS (RET-ROS) have been implicated in tissue damage during organ transplantation, whereas an RET-induced NAD+/NADH ratio decrease has been implicated in aging, age-related neurodegeneration, and cancer. In this review, we provide a historical account of the roles of ROS and oxidative damage in the pathogenesis of ischemic stroke, summarize the latest developments in our understanding of RET biology and RET-associated pathological conditions, and discuss new ways to target ischemic stroke, cancer, aging, and age-related neurodegenerative diseases by modulating RET.

Abstract 1382: Expression and intracellular translocation of cancer biomarkers in hepatocarcinoma cells induced by changes in mitochondrial metabolism

Abstract Hepatocarcinoma is one of the most common cancer types in the U.S. with limited treatment options and poor survival rate. Recently, cancer bioenergetics emerged as a promising source of targets to develop novel anticancer agents. The identification of biomarkers is essential for screening and validation of small molecules proposed as chemotherapeutic agents. In this regard, there is a great need for high-throughput/high-content methods, which require short and reproducible sample preparation. In this project, we used the erastin-like compound X1 in the well-characterized SNU-449 hepatocarcinoma cell line. Erastin initially enhances mitochondrial function and ROS formation leading to subsequent mitochondrial dysfunction. As readout, we employed directly and indirectly labeled fluorescent antibodies to visualize and quantify expression of proliferative and antiproliferative markers Ki-67 and cytoskeleton-associated protein 4/p63 (CKAP4/p63), as well as expression and intracellular translocation of the tumor suppressor protein p53. Using the Agilent BioTek Cytation 5 cell imaging multimode reader and Agilent BioTek Gen5 microplate reader and imager software, we successfully validated these markers and the corresponding antibodies as tools to study long-term effects of xenobiotics on mitochondrial bioenergetics. The method allows for fast and reliable screening of potential drug candidates. Acknowledgement: This work was supported in part by the Chan Zuckerberg Initiative, Silicon Valley Community Foundation, and the NIH S10 OD028663 to M.G. and by the U.S. National Institutes of Health (NIH/NCI) R01 CA184456, and South Carolina Translational Research Pilot Project UL1 TR001450-SCTR to E.N.M. Citation Format: Bradley R. Larson, Monika Gooz, Eduardo Maldonado. Expression and intracellular translocation of cancer biomarkers in hepatocarcinoma cells induced by changes in mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1382.

Multiple targets related to mitochondrial function unveiled by metabolomics and proteomics profiles of hearts from atrial fibrillation patients.

Background: The prominent mitochondrial metabolic changes of the atrium reportedly have significant impact on electrical signals and structural remodeling which play important roles in the occurrence and development of atrial fibrillation (AF). However, the mechanism is not completely known. Objective: This study was aimed to explore the mitochondrial metabolism reprogrammed in AF patients by integrating metabolomics as well as proteomics of human atrium tissues. Methods and Results: Left atrial tissue samples were harvested from 10 non-valvular AF patients and 10 matched samples from healthy donors for transplantation. In metabolomics analysis, 113 metabolites were upregulated and 10 metabolites were downregulated in AF, where multiple pathways related to mitochondrial energy metabolism were enriched. Correlation analysis between the differentially expressed proteins and metabolites identified several hub proteins related to mitochondrial function including Glycerol-3-phosphate dehydrogenase 2 (GPD2), Synemin (SYNM), Plectin (PLEC), with MCC score of 27, 17, 16, respectively, which have the most interactions with the dysregulated metabolites and ranked at the top in network string interactions scored by MCC method. All 330 differentially expressed proteins including 225 upregulated and 105 downregulated molecules were revealed and analyzed, which identified the downregulation of GPD2 (p = 0.02 and FC = 0.77), PLEC (p < 0.001 and FC = 0.71) and SYNM (p = 0.04 and FC = 0.76) in AF patients. Gene Set Variation Analysis (GSEA) showed mitochondrial metabolism-associated pathways including oxidative phosphorylation (NES: -1.73) and ATP biosynthetic process (NES: -2.29), were dramatically diversified in human AF. In GSVA, the expression levels of GPD2, PLEC, and SYNM were demonstrated to be associated with multiple metabolic pathways related to mitochondrial function (e.g., lipid metabolism and AMP activated protein kinase signaling) and cardiac structural and electrical remodeling (e.g., contractile fiber, ion homeostasis), which were proven vital in the development and maintenance of AF. Conclusion: In all, this study provides new insights into understanding the mechanisms of AF progression, especially the reprogramming mitochondrial metabolism, and identifies several genes related to mitochondrial function as novel targets for AF, which may be involved in the occurrence and development of AF.

The NAMPT Inhibitor FK866 in Combination with Cisplatin Reduces Cholangiocarcinoma Cells Growth.

It is well established that Cholangiocarcioma (CCA) drug resistance plays a crucial role in the spread and survival of cancer cells. The major enzyme in the nicotinamide-adenine dinucleotide (NAD+)-mediated pathways, nicotinamide phosphoribosyltransferase (NAMPT), is essential for cancer cell survival and metastasis. Previous research has shown that the targeted NAMPT inhibitor FK866 reduces cancer cell viability and triggers cancer cell death; however, whether FK866 affects CCA cell survival has not been addressed before. We show herein that NAMPT is expressed in CCA cells, and FK866 suppresses the capacity of CCA cells to grow in a dose-dependent manner. Furthermore, by preventing NAMPT activity, FK866 significantly reduced the amount of NAD+ and adenosine 5'-triphosphate (ATP) in HuCCT1, KMCH, and EGI cells. The present study's findings further show that FK866 causes changes in mitochondrial metabolism in CCA cells. Additionally, FK866 enhances the anticancer effects of cisplatin in vitro. Taken together, the results of the current study suggest that the NAMPT/NAD+ pathway may be a possible therapeutic target for CCA, and FK866 may be a useful medication targeting CCA in combination with cisplatin.